MO reports PR3 dephosphorylates TZ Xian-o Lv et al xpanded View igures igure V1. PR3 dephosphorylates and inactivates YP/TZ., Overexpression of tight junction proteins Pals1 () or LIN7 () has no effect on the phosphorylation of TZ on Ser89. HK293T cells were transiently transfected with H- Pals1 and Myc-LIN7 in the presence of TZ as indicated, and the cells were harvested for Western blot analysis., The PR complex components have no effects on TZ phosphorylation. H-aPK and P- PR6/ were transfected into HK293T cells, and cell lysates were harvested for Western blot analysis. Verification of PR3 sirn knockdown efficiency in 375 and T-47 cells. Samples were detected by real-time PR. The results are average SM of three independent experiments. V1 MO reports ª 2015 The uthors
Xian-o Lv et al PR3 dephosphorylates TZ MO reports igure V2. PR3 interacts with LTS1 and PP1. PR3 has no effect on the interaction between TZ and its kinase LTS1. HK293T cells were transfected with P-tagged TZ and L-tagged LTS1 in the presence or absence of H-PR3, and proteins were immunoprecipitated with L beads for Western blot analysis. PR3 does not affect the binding of P-TZ and its phosphatase PP1. The indicated constructs were transfected into HK293T cells, and proteins were immunoprecipitated with L beads for Western blot analysis. PR3 enhances the interaction of PP1 with endogenous LTS1. HK293T cells were transfected with L-tagged PP1 in the presence or absence of H-PR3, and proteins were immunoprecipitated with L beads for Western blot analysis., PR3 interacts with PP1 and LTS1. The indicated constructs were transfected into HK293T cells, and proteins were immunoprecipitated with L antibody. ell lysates were analyzed by Western blot as indicated. The interaction of PP1/PR3/LTS1 is regulated by cell density. H-PR3 was transfected into T-47 cells, immunoprecipitation with LTS1 antibody was performed, and the immunoprecipitated samples were used for Western blot analysis as indicated. LTS1 S909 mutant is insensitive to PR3 regulation. WT and S909 were cotransfected into LTS1/2 KO 293 cells with H-TZ. Western blot analysis was performed as indicated. ª 2015 The uthors MO reports V2
MO reports PR3 dephosphorylates TZ Xian-o Lv et al igure V3. Phosphorylation of PR3 at Ser144/Ser873 diminishes TZ dephosphorylation. S144 and S873 of PR3 diminish phosphorylation of TZ at Ser89. The indicated constructs were cotransfected with L-TZ into HK293T cells. Western blot analysis was performed as indicated. Immunofluorescence localization of PR3 WT, 2S mutant, and 2S mutant. MK cells stably expressing WT, 2S, or 2S of PR3 were stained with H antibody. The quantification is shown below. 2S mutant but not the WT and 2S mutant of PR3 decreases interaction between PP1 and endogenous LTS1. The indicated constructs were transfected into HK293T cells, and proteins were immunoprecipitated with L antibody. ell lysates were analyzed by Western blot. Wild type and 2S but not 2S mutant of PR3 increases the interaction between L-PP1 and endogenous LTS1. The indicated constructs were transfected into HK293T cells, and proteins were immunoprecipitated with L antibody. ell lysates were analyzed by Western blot as indicated. PR1 induces TZ dephosphorylation at Ser89. HK293T cells were transiently transfected with H-PR1 and L-TZ, and the cells were harvested for Western blot analysis. Knockdown of PR1 increases the phosphorylation of TZ and inhibits the expression of T. 293T cells were transfected with sirn targeting PR1, the phosphorylation of TZ was detected by Western blot, and the expression of T was detected using real-time PR. Knockdown of PR1 but not PR3 decreases the phosphorylation of MST1/2 at Thr183/180. T-47 cells were transfected with the indicated sirns, and cells were harvested at high density for Western blot analysis. The knockdown efficiencies were determined by real-time PR on the right. Knockdown of PR1 increases the phosphorylation of MST1/2 at Thr183/180 slightly. T-47 cells were transfected with the indicated sirns, and cells were harvested at low density for Western blot analysis. V3 MO reports ª 2015 The uthors
Xian-o Lv et al PR3 dephosphorylates TZ MO reports igure V4. (continued) ª 2015 The uthors MO reports V4
MO reports PR3 dephosphorylates TZ Xian-o Lv et al igure V4. Knockdown of PR3 reduces the function of YP/TZ. WT and 2S mutant, but not 2S mutant, of PR3 rescue the expression of target genes inhibited by PR3 knockdown. T-47 cells with stable knockdown and overexpression of WT and 2S and 2S mutants of PR3 were used as indicated. The expression of target genes was analyzed by real-time PR. The efficiency of endogenous PR3 knockdown is shown above, and the expression of exogenous H-PR3 was verified by Western blot. The results are average SM of three independent experiments. Knockdown of PR3 increases -cadherin expression and decreases N-cadherin expression at the mrn level in T-47 cells at low cell density. PR3-knockdown T-47 cells were analyzed by qpr. The results are average SM of three independent experiments. Knockdown of PR3 inhibits T-47 cell growth which can be rescued by overexpression of YP2-5S. The results are average SM of 3 independent experiments., Overexpression of TZ-4S () and YP2-5S () PR3-knockdown T-47 cell. It was verified by Western blot and real-time PR. Knocking down TZ inhibits the growth of T-47 cells overexpressing PR3. rowth curves of TZ knockdown T-47 cells overexpressing PR3 were determined. ell lysates were analyzed by Western blot to verify H-PR3 and TZ expression. The results are average SM of 3 independent experiments. WT and 2S mutant, but not 2S mutant, of PR3 rescue colony formation inhibited by PR3 knockdown. T-47 stable cell lines (5 10 3 cells) were cultured in 6-well plates for 3 weeks. olonies were visualized by crystal violet staining and counted. The results are average SM of three independent experiments. **P < 0.01. V5 MO reports ª 2015 The uthors